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| Status |
Public on Nov 08, 2012 |
| Title |
Conserved DNA methylation patterns in healthy blood cells and extensive changes in leukemia measured by a new quantitative technique. |
| Organism |
Homo sapiens |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
We describe a simple method, Digital Restriction Enzyme Analysis of Methylation (DREAM), based on sequential DNA digestion with a pair of methylation-blocked and methylation-tolerant neoschizomeric restriction enzymes SmaI/XmaI followed by end repair and ultra-deep sequencing. DREAM provides information on 160,000 unique CpG sites of which 39,000 are in CpG islands, and 33,000 are at transcription start sites (-1 kb to +1 kb) of 13,139 RefSeq genes. We compared DNA methylation values in white blood cells from 4 healthy individuals and found them to be remarkably uniform. Interindividual differences >30% were observed only at 227 of 28,331 (0.8%) of autosomal CCCGGG sites covered by 100+ sequencing reads. Similarly, differences at only 59 sites were observed between the cord and adult blood. Conserved methylation patterns in healthy blood cells contrasted with extensive changes affecting 18-40% of CpG sites in leukemia. The method is cost effective, quantitative (r2=0.93 when compared to bisulfite pyrosequencing), reproducible (r2=0.997), and can detect differences >25% with false positive rate <0.001. Accurate analysis of changes in DNA methylation will be useful in quantifying epigenetic effects of environment and nutrition, correlating developmental epigenetic variation with phenotypes, understanding epigenetics of cancer and chronic diseases, measuring the effects of drugs on DNA methylation or deriving new biological insights into mammalian genomes.
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| Overall design |
Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the DNA methylation profiles of healthy white blood cells from cord blood and adult blood, acute myeloid leukemia bone marrow, and two leukemia cell lines (HEL and K562). In this approach, genomic DNA is sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represent methylation status of the CCCGGG sites. These end sequences are analyzed by next-generation sequencing, and thereafter the methylation status at individual CCCGGG sites across the genome can be determined.
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| Contributor(s) |
Jelinek J, Liang S, Lu Y, He R, Ramagli LS, Shpall EJ, Estecio MR, Issa JP |
| Citation(s) |
23075513 |
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| Submission date |
Jul 31, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Jaroslav Jelinek |
| E-mail(s) |
[email protected]
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| Phone |
215-707-7312
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| URL |
http://www.temple.edu
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| Organization name |
Temple University
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| Department |
Fels Institute
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| Lab |
Issa Lab
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| Street address |
3307 North Broad St
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19140 |
| Country |
USA |
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| Platforms (2) |
| GPL9115 |
Illumina Genome Analyzer II (Homo sapiens) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
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| Samples (13)
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| Relations |
| BioProject |
PRJNA171709 |
| SRA |
SRP014638 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE39787_RAW.tar |
37.8 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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